1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions for the identification and selection of insertional mutants.
2. Description of Related Art
Mutants are powerful tools in the investigation of physiological, developmental, and cell biological processes. Starting with a phenotypic mutant generated by chemical mutagenesis, it is possible to use a genetic map-based strategy to clone a gene (Arondel et al., 1992). Mutations derived from insertional mutagenesis are particularly useful in that they provide "tagged" copies of the mutated gene which may readily be cloned (Yanofsky et al., 1990). However, molecular genetic techniques have advanced such that today most genes are cloned and sequenced long before their function is characterized genetically (Newman et al., 1994). For many genes, phenotypic screens are not available, and mutations which cause lethality remain undetectable. What has been missing is a simple and reliable strategy to go from a gene or protein sequence to the identification of specific mutants.
One solution to problems associated with mutant identification was to use the polymerase chain reaction (PCR) to screen for P-element mutations in sequenced genes of Drosophila (Ballinger et al., 1989; Kaiser et al., 1990). This approach also enhanced the genetics of Caenorhabditis (Rushforth et al., 1993; Zwaal et al., 1993), where transposable element mutations are now commonly isolated for known gene sequences. In these systems, transposon-induced mutations are isolated for known gene sequences by the general strategy known as "site-selected" mutagenesis. Basically, the method relies on the power of PCR to amplify a collection of specific junction fragments between an inserted element and a known target gene sequence from large pools of randomly inserted elements. One primer is used which is homologous to the end of the inserted element with its 3' end facing outward and one primer within the target gene is used to amplify the sequences at the junction of the insertion. In plants, similar approaches have been used to identify insertion mutations in Petunia, using the transposon dTph1 (Koes et al. 1995), and in Arabidopsis using collections of T-DNA transformed lines (Krysan et al., 1996; Mckinney et al., 1995). In Krysan et al. (1996), 9100 independent T-DNA-transformed Arabidopsis lines (averaging 1.4 insertions per genome) were subjected to site-selected mutagenesis and 17 T-DNA insertions within 63 genes were identified.
While techniques based on the gene-specific amplification of insertional junctions have been useful in the isolation of a number of mutants, they have had limited success in applications toward large-scale genomic investigations. The need for individual amplifications of each gene being investigated represents a significant hindrance when seeking to identify more than a small number of insertional mutants. There is, therefore, a great need in the art for a method by which large numbers of insertional mutants may be rapidly and efficiently identified.